Area of the Art
The present invention is in the area of pluripotent stem cells and more particularly deals with a method to differentiate a vascular network from stem cells.
Description of the Background Art
Perhaps the greatest roadblock to the success of tissue regenerative therapies is the establishment of a functional microvascular network to support tissue survival and growth (Discher et al., 2009). Microvascular construction or regeneration depends on endothelial morphogenesis into a three-dimensional, tubular network followed by stabilization of the assembling structures by recruited pericytes (Hanjaya-Putra et al., 2011; Stratman et al., 2009a). To create such a construct for therapeutic applications, patient-derived ECs and pericytes must be incorporated into a synthetic matrix, which confers the advantage to control and modulate vascular morphogenesis and simultaneously represents a clinically-relevant construct in which to deliver the engineered microvascular networks to in vivo environments (Vunjak-Novakovic and Scadden, 2011).
Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs), offer the opportunity to derive EVCs from the same source, the latter of which offers patient specificity. Various cell markers have been proposed to identify vascular precursors (of ECs and pericytes) from differentiating hPSCs including CD34 (Ferreira et al., 2007; Park et al., 2010), KDR/VEGFR2 (Yang et al., 2008), and apelin receptor (Vodyanik et al., 2010). Purification of such progenitors is required from an uncontrolled differentiated cell population (i.e. via embryoid body [EB] formation or co-culture on mouse feeder layer) through marker enrichment or selection through genetic manipulation. Importantly, none of these derived cells have been demonstrated to self-assemble into functional microvasculature containing both ECs and pericytes.
Current approaches for the differentiation of hPSCs toward the vascular lineage build on the notion that a purified, single derivative—either a progenitor or matured cell type—is obligatory for the generation of functional vasculature. These approaches stem from the necessity to eliminate differentiation to undesirable lineages as well as to better understand the development of the vasculature. Indeed, from this body of work, it has become apparent that various cell markers and biochemical cues can be used to guide differentiation and derive functional ECs (Drukker et al., 2012; Ferreira et al., 2007; James et al., 2010; Wang et al., 2007), vascular smooth muscle cells (Drukker et al., 2012; Ferreira et al., 2007; Wanjare et al., 2012) and pericytes (Dar et al., 2011). Here we disclose a new conceptual approach in which the cells of the microvasculature are derived in a single, bipotent type population. The developed protocol employs a monolayer culture and avoids an EB intermediate and sorting, thereby ensuring reproducibility and clinical applicability. The derived bipotent population is able to work synergistically to recreate the tissue. Thus, we harness intrinsic tissue-level differentiation and self-assembly capabilities toward the translational realization of hPSCs. This new paradigm could prove useful for the construction of other multicellular tissues for regeneration.
The current disclosure demonstrates that hPSCs can be induced to differentiate into early derivatives of the vascular lineage (i.e. EVCs) that comprise the microvascular architecture without a specific differentiation-inducible feeder layer, EB formation, or genetic manipulation, and that such EVCs can mature into ECs and pericytes and can self-assemble to form functional vascular networks in an engineered matrix.
The ability to derive a multi-cell type population, which is then leveraged to form physiologically- and clinically-relevant vascular networks that are functionally perfused in vivo, is dependent upon activation of Notch signaling. Inhibition of Notch signaling promoted EC differentiation as depicted via VEcad enrichment. This discovery provides the groundwork for future studies into the importance of Notch signaling in in vitro vascular co-differentiation strategies. Our novel bicellular constructs represent a fundamental advancement to the future of cell-based therapies
The balance between commitment and plasticity of the EVCs specifically within the vascular lineage allows for vascular fate and functional network maturation. This controlled system is reproducible, generates physiologically relevant vascular networks in implantable matrices, and thus presents the next fundamental step toward patient-specific engineered tissue with clinically translatable potential.